Live attenuated vaccines are long known to induce superior immune protection and lasting immune memory, and with single dose administration in most cases. When compared to their dead counterparts, live vaccines induce a distinct and far more vigorous immune response, triggering an alert mode not warranted for dead microorganisms. But despite the efficacy of live vaccines, several factors have contributed to their lack of popularity. Chief among these are concerns over their safety as well as difficulty and increased cost in their delivery and preservation especially to impoverished areas in developing countries. Understanding the molecular basis for the efficacy of live vaccines would alleviate these concerns, and provide a means to target the relevant pathways that induce optimal protective immunity. The work we propose here is poised to significantly enhance our understanding of the basic immune mechanisms behind the superior efficacy of live attenuated vaccines. We began our work with the hypothesis that innate immune cells sense the most fundamental characteristic of microbial infectivity, microbial viability itself, and activate an immune response tailored to eradicate the infectious threat, and importantly regardless of the presence of specialized factors that regulate microbial virulence. We identified the signature molecule, prokaryotic messenger RNA, which alerts professional phagocytic cells of the innate immune system to the presence of viable microbes in sterile tissues. We identified discrete innate immune responses triggered by the recognition of bacterial viability. We have dissected the innate immune pathways mediating these responses, and determined the identity of the critical Toll-like receptor signaling adaptor that orchestrates these responses. Our goal here is t identify how the alert mode triggered by bacterial viability impacts the subsequent adaptive immune response. With identity of the viability-sensing pathways and the immunostimulatory properties of the bacterial mRNA in hand, we will determine the role of individual players within these pathways in the evolution of the immune response. Unlike previous studies that have used adjuvants and haptenated proteins, we will focus on antigens presented within the context of a bacterial infection. We will investigate both the T cell and B cell immune response beginning first with understanding the nature of the CD4 T cell response because of its critical link to the T cell-dependent antibody response. We will next dissect the antibody response to both T cell-dependent and T cell-independent antigens expressed by both live and dead bacteria. We will define how deficiency in the pathways sensing bacterial viability alters the immune response, depriving it of the factors critical for long lasting protective immunity. Our studies have the high potential to reveal the mechanisms behind the superior protection induced by live vaccines, and to create new adjuvants and immunization strategies that combine the efficacy of live vaccines with the safety of dead vaccines. PUBLIC HEALTH RELEVANCE: Live attenuated vaccines are notoriously superior to dead vaccines, yet the reasons for this remain unknown. We identified discrete innate immune responses triggered specifically by live bacteria, and our intention here is to understand how these responses work together to induce protective adaptive immunity. Our studies have the potential to create immunization strategies that combine the superior protection of live vaccines with the safety of dead vaccines.